Summary Apoptosis is crucial for proper development and function of cell populations in tissues, and its dysregulation is of major relevance for degenerative diseases and cancer. The critical step in triggering apoptosis is the permeabilization of the mitochondrial outer membrane (MOMP). This process is tightly regulated by the Bcl-2 family of proteins, which is subdivided into pro-apoptotic (e.g., Bax), anti-apoptotic (e.g., Bcl-xL), and BH3- only regulator proteins (e.g., Bid). Despite recent advances in the characterization of Bcl- 2 proteins, the field lacks a mechanistic understanding of the protein?protein and protein? lipid interactions that mediate MOMP. Such knowledge would be essential for setting the stage for the future development of therapeutic strategies aimed at either suppressing or activating apoptosis. The proposed project is aimed at deciphering the pathways of membrane insertion and refolding of the Bax/Bid/Bcl-xL regulatory triad. Site-specific labeling in combination with a battery of fluorescence (including various types of steady- state and lifetime quenching and FRET) and electron paramagnetic resonance approaches, complemented by Molecular Dynamics computer simulations, will be utilized to obtain structural, dynamic and thermodynamic information necessary for deciphering the mechanism of physiological function. By gaining new insights into molecular mechanisms of protein?protein and protein?lipid interactions in the Bax/Bcl-xL/Bid regulatory triad, we expect to provide a clearer map of the molecular pathways controlling MOMP. In this supplement we request the funds to replace our obsolete and not serviceable 15-year old Fluorolog-3 fluorometer with the state-of-the-art modern version of the instrument. Maintaining fluorescence capabilities is absolutely necessary for the implementation of the funded parent research proposal. In addition, the requested upgrade includes iHR320-FAS imaging spectrometer and Hi-Tech SFA-20 Stopped Flow accessory, which will enhance the original research plan by providing capabilities for kinetic characterization of the bilayer insertion and refolding transitions in apoptotic regulators; and for identifying critical intermediate states.